Mucus is one of the human body's primary defenses against pathogens. Mucus is built from proteins with hydrophobic and hydrophilic domains, where the hydrophobic domains associate together to form physical gels. Size exclusion and ionic repulsion restrict most molecules such as viruses from penetrating the gel, see FIG. 1. In many cases, mucus provides a selective filter that can allows nutrients and information to pass while keeping pathogens and toxins out. Mucus can significantly decrease the bioavailability of medicinal drugs for example. Mucins are a class of glycoproteins that are categorized by their amino acid backbone composition, glycosylation pattern, and typical location within the body. Their multiple functions are shown in FIGS. 2A and 2B. Mucins, the main gel-forming constituents of the mucus, additionally demonstrate the effect of specific binding to glycosyl sequences as a mechanism to regulate the passage of pathogens. Therefore, pharmaceutical companies are very interested in testing drug candidates to determine mucus permeability. The defense industry views mucins as a possible method for combating exogenous biological threat.
However, acquiring large quantities of mucus, such as mucins, can be challenging. A common source of mucins is obtaining them by scraping pig stomachs; however, this process typically yields mucins on the order of micrograms. Further, mucins are high molecular weight polymers that range from several hundred thousand to several million Daltons. The high molecular weight and glycosylation of mucins make them very challenging to synthesize via molecular biology. In nature, mucins are synthesized as a lightly glycosylated, thiol-reduction-resistant precursor in the golgi apparatus. This precursor is subsequently glycosylated in the endoplasmic reticulum and golgi apparatus and then further modified after secretion outside of the cell. Known mucin mimics are typically synthesized either as short glycosylated oligomers that are polymerized resulting in low-molecular weight mimics or expressed as proteins that are glycosylated with expensive enzymes resulting in a low degree of glycosylation.
Provided herein is a series of brush proteins that mimics the variable number of tandem repeats (VNTR) of respiratory mucins that have the capability to be chain-extended through disulfide coupling and the use of a bioconjugation technique to post-translationally mass functionalize proteins. In nature, enzymes functionalize threonines and serines via glycosylation. As reproducing this process in vitro is both expensive and challenging, we used diazonium coupling based tyrosine modification chemistry that is orthogonal to cysteine based chain extension functionalization. Diazonium coupling is typically used to bioconjugate proteins on a single location. Here, we describe the use of this chemistry to mass functionalize a protein. This method of economically mimicking post-translational modification enables the production of high molecular weight and densely functionalized mucin mimetic materials.
There exists a need for mimics of mucins that retain their physical and functional characteristics. The proteins described herein provide a series of mucin mimics that are useful in developing and testing, for example, pharmaceutical drug metabolic properties.